Genome-Wide Identification of the SPP/SPPL Gene Family and BnaSPPL4 Regulating Male Fertility in Rapeseed (Brassica napus L.)

Abstract

Signal peptide peptidase (SPP) and its homologs, signal peptide peptidase-like (SPPL) proteases, are members of the GxGD-type aspartyl protease family, which is widespread in plants and animals and is a class of transmembrane proteins with significant biological functions. SPP/SPPLs have been identified; however, the functions of SPP/SPPL in rapeseed (Brassica napus L.) have not been reported. In this study, 26 SPP/SPPLs were identified in rapeseed and categorized into three groups: SPP, SPPL2, and SPPL3. These members mainly contained the Peptidase_A22 and PA domains, which were distributed on 17 out of 19 chromosomes. Evolutionary analyses indicated that BnaSPP/SPPLs evolved with a large number of whole-genome duplication (WGD) events and strong purifying selection. Members are widely expressed and play a key role in the growth and development of rapeseed. The regulation of rapeseed pollen fertility by the BnaSPPL4 gene was further validated through experiments based on bioinformatics analysis, concluding that BnaSPPL4 silencing causes male sterility. Cytological observation showed that male infertility caused by loss of BnaSPPL4 gene function occurs late in the mononucleate stage due to microspore dysplasia.

Keywords: Brassica napus L.; SPP/SPPL gene family; expression profile; male sterility; pollen.

Figure 1

Phylogenetic analysis of SPP/SPPL family in B. napus, B. oleracea, B. rapa, and A. thaliana, classifying 57 SPP/SPPLs into three subgroups: SPP, SPPL2, and SPPL3. Red triangles represent 6 SPP/SPPLs in Arabidopsis, green circles represent 26 SPP/SPPLs in B. napus, blue boxes represent 12 SPP/SPPLs in B. oleracea, and orange pentagrams represent 13 SPP/SPPLs in B. rapa.

Figure 2

Analysis of gene structure, motif, and domains of BnaSPP/SPPLs and ATSPP/SPPLs: (A) phylogenetic tree; (B) conserved motifs; (C) domains; (D) introns, exons, and UTRs, where introns are marked with black horizontal lines.

Figure 3

The 26 BnaSPP/SPPLs are located on the chromosomes; the length of each chromosome is shown in Mb, the chromosomal number is shown on the left side of the chromosome, and the BnaSPP/SPPLs are highlighted in red.

Figure 4

Circular representations of BnaSPP/SPPL chromosomal dispersal and inter-chromosomal interactions, as well as synteny analysis of SPP/SPPL genes in B. rapa, B. oleracea, B. napus, and Arabidopsis. (A) Analysis of syntenic relationships between BnaSPP/SPPL paralog pairs; within the schematic image, duplicate pairs of BnaSPP/SPPLs are shown by red lines. (B) Analysis of syntenic relationships between BraSPP/SPPLs, BolSPP/SPPs, BnaSPP/SPPs, and ATSPP/SPPs, as shown by gray lines.

Figure 5

Ka/Ks values of BnaSPP/SPPL paralog pairs. Blue lines are Ka values, yellow lines are Ks values, and red lines are Ka/Ks. The X-axis and Y-axis indicate paralog pairs and Ka, Ks, and Ka/Ks values, respectively.

Figure 6

Expression pattern analysis of BnaSPP/SPPLs in different tissues. (A) Expression patterns of BnaSPP/SPPLs from the BnIR, scale bar is Log2 (TPM)-normalized expression, and blue denotes low expression and red denotes strong expression; the axes indicate BnaSPP/SPPLs the and phylogenetic tree. (B) RT-qPCR validation of BnaSPP5, BnaSPP6, BnaSPPL4-3, BnaSPPL3-4, and BnaSPPL4-4 in different tissues.

Figure 7

Construction of vector and detection of gene transcription level in transgenic rapeseed. (A) Schematic diagram of RNAi vector construction, and the expression vector was pFGC5941. (B) Gene transcription level of transgenic rapeseed; data are shown as mean ± SE (n = 3). (C) Statistical analysis of the size of petals, sepals, stamens, and pistil of transgenic rapeseed and WT; data are shown as mean ± SE (n = 20). The symbols ** indicate a statistically significant deviation from WT at p < 0.01 probability levels.

Figure 8

Photographic observation of transgenic rapeseed and wild-type (WT) phenotype: (A) phenotype of whole-plant rapeseed; (B) inflorescence of rapeseed; (C) the pollen was colored with carmine acetate stain solution (bars for 200 µm); (D) the petals, sepals, stamens, and pistil of rapeseed were photographed and observed.

Figure 9

Paraffin sectioning cross-sections: fertile (A–I) and sterile (J–O); (A,B,J,K) sporogenous cell stage; (C) pollen mother cell stage; (D) premeiosis stage; (E) tetrad stage; (F,L–N) late uninucleate microspore stage; (G,H) bicellular pollen stage. Sp, sporogenous; PMC, pollen mother cell; Tds, tetrads; Msp, microspores; PG, pollen grain; T, tapetum. (A,J) bars for 100 µm, magnifications for 200. (B–I,K–O) bars for 50 µm, magnifications for 400.